EXCHANGE 


An  Experimental   Study 

of 
Certain   Basic   Ammo   Acids 


DISSERTATION 

SUBMITTED  IN  PARTIAL  FULFILMENT   OF   THE   REQUIRE- 
MENT FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 
IN  THE  FACULTY  OF  PURE   SCIENCE, 
-:   COLUMBIA  UNIVERSITY 


BY 

MARY  L.  CALDWELL,  A.B.,  A.M. 

New  York  City 
1921 


The  Jackson  Prc«s, 
1921 


An  Experimental  Study 

of 
Certain  Basic   Ammo  Acids 


DISSERTATION 

SUBMITTED  IN  PARTIAL  FULFILMENT  OF  THE   REQUIRE- 
MENT FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 
IN  THE  FACULTY  OF  PURE  SCIENCE, 
COLUMBIA  UNIVERSITY 


BY 

MARY  L.  CALDWELL,  A.B.,  A.M. 

New  York  City 
1921 


The  Jacksoa  Press,  Kingston 
1921 


ACKNOWLEDGMENTS 

This  investigation  was  undertaken  at  the  suggestion  of  Professor 
H.  C.  Sherman,  and  was  carried  out  under  his  direction.  The 
author  wishes  to  express  to  Professor  Sherman  her  appreciation 
of  his  advice  and  encouragement  received  throughout  this  work. 

The  author  also  wishes  to  thank  Professor  A.  W.  Thomas  for 
many  helpful  suggestions. 

M.  L.  C. 


AN  EXPERIMENTAL  STUDY  OF  CERTAIN  BASIC 
AMINO  ACIDS 

The  object  of  this  investigation  was  to  study  the  influence  of 
certain  basic  amino  acids  upon  the  hydrolysis  of  "soluble"  starch 
by  purified  pancreatic  amylase.  And  since,  at  the  time  the  inves- 
tigation was  started,  it  was  impossible  to  buy  arginine  and  his- 
tidine,  it  was  decided:  First,  to  prepare  them  in  a  form  suitable 
for  the  enzyme  work ;  second,  to  study  their  action  with  the  enzyme. 

PREPARATION  OF  ARGININE  AND  HISTIDINE 

The  preparations  of  arginine  and  histidine  were  obtained  from 
commercial  gelatin  and  casein  by  the  method  first  worked  out  by 
Kossel  and  Kutscher1  in  1900-1901.  This  method  is  based  on  the 
separation  of  histidine  and  arginine  from  the  other  amino  acids 
present  in  the  hydrolysis  mixture  by  means  of  their  silver  salts, 
and  then  from  each  other  by  the  difference  in  solubility  of  these 
salts  in  neutral  and  in  strongly  alkaline  solutions.  This  method  has 
since  been  modified  and  improved  by  several  workers — Kossel  and 
Patten,2,  Steudel,3  Kossel  and  Pringle,4  Weiss,5  Osborne,  Leaven- 
worth  and  Brautlecht,6 — and  has  been  summarized  by  Plimmer.7 

In  carrying  out  the  preparation  it  was  found  that  this  method, 
with  its  modifications,  presented  difficulties;  being  vague  and  inde- 
finite in  places,  and  conflicting  directions  being  given.  An  attempt 
was  therefore  made  to  standardize  the  process  more  fully.  The 
preparation  was  as  follows : — 

Hydrolysis  and  Estimation  of  Protein. — About  100  grams  of 
protein  were  hydrolyzed  by  boiling  with  a  mixture  of  300  grams 
of  concentrated  sulfuric  acid  and  600  grams  of  water  under  a  reflux 
condenser  for  24  hours  in  an  oil  bath  which  was  kept  at  about 
105 °C.  A  biuret  test  at  this  point  was  always  negative,  indicating 
complete  hydrolysis  of  protein  to  amino  acids.  The  solution  thus 

iZ.  physiol.  Chem.,  (1900-1901)  31,  165-214. 

2Ibid   (1903)  38,  39-45. 

sibid   (1902-1903)   37,  219-220;   (1905)  44,  157-158. 

*Ibid  (1906)  49,  301-321. 

'Ibid  (1907)  52,  107. 

«Am.  Jour.  Physiol.   (1908)  23,  180-200. 

7The  Chemical  Constitution  of  the  Proteins,  Part  I  (1917),  55-63. 


502089 


obtained  was  then  filtered,  if  not  clear,  and  made  up  to  1  liter  with 
water.  Nitrogen  determinations  were  made  in  5  cc.  portions  by 
the  Kjeldahl  method,  and  from  these  determinations  the  total 
amount  of  protein  in  the  mixture  was  calculated,  since  the  per- 
centage of  nitrogen  in  the  pure  protein  was  known. 

Removal  of  Sulfuric  acid. — Most  of  the  sulfuric  acid  was  re- 
moved from  the  solution  by  adding  a  little  less  than  the  calculated 
amount  of  barium  hydroxide.  This  was  added  in  the  form  of  a 
hot  solution,  slowly,  with  constant  stirring,  until  the  reaction  of 
the  main  solution  was  alkaline  to  Congo-red,  but  still  acid  to 
litmus.1 

At  this  point  the  recommendations  of  previous  investigators 
were  conflicting,  and  sometimes  vague.  The  use  of  barium  hy- 
droxide, in  the  form  of  a  hot  or  a  cold  solution,  or  in  the  form  of 
a  solid,  has  been  advocated,  and  it  was  to  be  added  "until  the  re- 
action is  only  faintly  acid  and  almost  the  whole  of  the  sulfuric  acid 
is  precipitated  as  barium  sulfate." 

The  use  of  the  hot  solution  was  found  best.  The  precipitate 
formed  is  easier  to  filter,  and  the  danger  of  decomposing  the  ar- 
ginine  present  by  local  excess  of  alkali  is  decreased.  The  use  of 
Congo  red  paper  was  adopted  as  indicating  the  correct  concentration 
of  hydrogen-ion  at  this  point. 

The  precipitate  of  barium  sulfate  thus  formed  was  carefully 
washed  by  stirring  up  with  water  in  a  mortar  and  filtering  by  suc- 
tion. This  was  repeated  until  the  filtrate  gave  no  precipitate  with 
a  solution  of  phosphotungstic  acid.  Usually  three  washings  were 
sufficient. 

Evaporation. — The  filtrate  and  washings  were  evaporated  ir> 
vacuo  at  70° C.  and  made  up  to  1  liter.  A  determination  of  nitrogen 
was  made  with  5  cc.  portions  of  this  solution.  This  gave,  by  dif- 
ference, the  amount  of  nitrogen  removed  in  the  first  barium  sulfate 
precipitate.. 

This  leads  to  another  inconsistent  point  in  the  methods  as  pre- 
viously described.  It  is  recommended  that  the  above  evaporation 
of  the  slightly  acid  solution  be  carried  out  in  vacuo,  while  later  on 
in  the  processes,  alkaline  solutions  are  evaporated  in  open  evapo- 
rating dishes  at  100 °C.  This  is  the  case  in  the  removal  of  ammonia 

JDakin:  J.  Biol.  Chem.  (1920)  44,  499-529. 


from  the  solution  made  alkaline  with  barium  carbonate,  and  also 
in  the  Kossel  and  Pringle  modification  for  separating  the  histidine 
silver  compound  from  the  arginine  silver  compound,  by  warming 
and  then  boiling  the  solution  with  barium  carbonate. 

Since  it  had  been  found  best  to  carry  out  some  of  the  evapo- 
rations in  vacuo,  it  was  decided  that  it  would  be  more  consistent  to 
carry  out  all  evaporations  in  vacuo,  at  70°  C.  This  decision  was 
found  to  be  justified  by  experiment,  the  difference  being  especially 
noticeable  in  the  yield  of  arginine,  as  would  be  expected.  In  two 
experiments  with  casein,  one  conducted  partly  in  vacuo  and  partly 
in  open  evaporating  dishes,  and  the  other  in  vacuo  entirely,  the 
yields  of  nitrogen  in  the  arginine  fractions  were  0.35  per  cent,  and 
1.1  per  cent  of  the  protein  respectively. 

In  the  vacuum  distillations  three-liter  flasks  were  found  con- 
venient, as  the  larger  flasks  made  evaporation  more  rapid,  and  less- 
ened the  danger  of  loss  from  foaming.  An  ordinary  water-pump 
was  used,  and  a  pressure  of  30  mm.  or  less  was  maintained.  Oil 
baths  were  found  to  be  better  than  water  baths,  as  the  temperature 
remained  more  constant,  and  trouble  by  evaporation  of  the  water 
was  eliminated. 

Estimation  of  Ammonia. — The  ammonia  formed  in  the  hy- 
drolysis of  the  protein  was  estimated  by  distilling  50  or  100  cc. 
portions  of  this  solution  with  a  slight  excess  of  magnesium  oxide 
paste  (litmus),  collecting  the  distillate  in  standard  acid,  and  titrat- 
ing, using  methyl  red  as  indicator.  The  magnesium  oxide  was  first 
freed  from  ammonia  by  boiling  with  water. 

Removal  of  Ammonia. — The  ammonia  was  removed  from  the 
remainder  of  the  solution  by  evaporating  with  a  slight  excess  of 
barium  carbonate.  The  separate  solutions  were  united  and  filtered, 
and  the  barium-magnesium  precipitate  carefully  washed  as  before. 
The  excess  of  barium  was  removed  by  dilute  sulfuric  acid,  and  the 
resulting  precipitate  again  carefully  washed.  The  filtrate  and  wash- 
ings were  evaporated  down,  made  up  to  one  liter,  and  5  cc.  portions 
used  for  Kjeldahl  determinations.  In  this  way  the  amount  of 
nitrogen  removed  in  the  second  precipitation  could  be  determined. 

Comparison  of  the  results  obtained  in  the  different  hydrolysis 
mixtures  shows  that  the  amounts  of  nitrogen  removed  by  these 
precipitations  were  quite  constant,  and  were  a  little  lower  for  gelatin 
than  for  casein.  Given  in  percentage  of  total  nitrogen  of  the  hy- 
drolysis mixtures,  they  were:  for  casein,  "first  barium  sulfate  pre- 


precipitate,"  9.76  per  cent  and  8.86  per  cent,  "second  barium  sulfate 
precipitate,"  2.65  per  cent  and  2.04  per  cent;  for  gelatin,  "first 
barium  sulfate  precipitate/'  6.87  per  cent  and  5.79  per  cent,  "second 
barium  sulfate  precipitate,"  0.21  per  cent  and  0.54  per  cent. 

Precipitation  of  Arginine  and  Histidine. — The  cold,  slightly 
acid  solution  was  treated  with  a  hot,  saturated  solution  of  silver 
sulfate,  which  was  added  slowly  with  stirring  until  the  solution  con- 
tained sufficient  silver  to  give  a  yellow-brown,  not  a  white  or  pale 
yellow,  precipitate,  on  removing  a  drop  and  testing  it  with  a  drop 
of  barium  hydroxide  solution  on  a  test  plate.  The  brown  precipi- 
tate should  form  immediately  to  show  excess  of  silver,  as  the  white 
silver  histidine  and  arginine  precipitate  formed  will  decompose  on 
standing  a  few  minutes  and  turn  brown. 

In  order  to  be  able  to  follow  the  nitrogen  content  of  the  dif- 
ferent fractions,  silver  sulfate  rather  than  silver  nitrate  must  be  used 
here ;  but  owing  to  its  slight  solubility,  the  operation  requires  much 
time,  and  more  water  must  constantly  be  added  to  the  mixture,  since 
the  silver  sulfate  keeps  separating  out  as  the  solution  cools.  As 
soon  as  enough  silver  sulfate  had  been  added,  the  cold  solution  was 
saturated  with  finely  powdered  barium  hydroxide  in  order  to  pre- 
cipitate the  silver  compounds  of  histidine  and  arginine.  Since  the 
barium  hydroxide,  even  when  finely  powdered,  dissolves  slowly,  an 
error  may  be  introduced  here,  resulting  in  an  incomplete  precipita- 
tion of  the  arginine,  unless  care  is  taken  that  the  solution  is  actually 
saturated.  This  precipitate  was  filtered  off  and  carefully  washed  by 
stirring  up  several  times  in  a  mortar  with  barium  hydroxide  solu- 
tion. 

Nitrogen  in  the  Lysine  and  Monoamino  Acid  Fraction. — The 
filtrate  from  the  above  precipitate  was  acidified  with  sulfuric  acid, 
freed  from  silver  with  hydrogen  sulfide,  and  the  filtrates  and  wash-, 
ings  evaporated  down  to  a  definite  volume,  so  that  nitrogen  deter- 
minations could  be  made  in  suitable  aliquot  portions.  In  this  way 
the  distribution  of  nitrogen  in  the  above  separation  could  be  fol- 
lowed. 

Decomposition  of  Silver  Compounds  of  Arginine  and  Histi- 
dine.— The  precipitate  of  the  silver  salts  of  histidine  and  arginine 
obtained  above  was  suspended  in  water  made  acid  with  a  known 
amount  of  sulfuric  acid,  and  decomposed  with  hydrogen  sulfide. 
The  precipitate  of  silver  sulphide  and  barium  sulfate  formed  was 


— 7— 

carefully  washed.  It  was  found  that  unless  special  care  was  taken 
here,  silver  was  carried  over  in  the  filtrate.  The  thorough  decompo- 
sition of  the  silver  compounds  of  histidine  and  arginine  was  there- 
fore made  certain  by  saturating  the  filtrate  and  washings  also  with 
hydrogen  sulfide.  This  precaution  was  again  borne  in  mind  when 
decomposing  these  silver  compounds  separately  by  hydrogen  sulfide 
in  acid  solution. 

The  filtrate  and  washings  were  evaporated  down  to  remove 
hydrogen  sulfide,  made  up  to  1  liter  and  Kjeldahl  determinations 
made  in  suitable  aliquot  portions.  Thus  the  amount  of  nitrogen  in 
the  arginine  and  histidine  fraction  was  obtained. 

Separation  of  the  Histidine  Fraction. — Several  methods  have 
been  recommended  for  separating  the  histidine  from  the  arginine  in 
the  above  mixture. 

(a)  In  the  original  Kossel  method1  the  separation  is  based 
entirely  on  the  difference  in  the  solubility  of  the  silver  compounds 
of  histidine  and  arginine  in  neutral  and  in  strongly  alkaline  solu- 
tions respectively.    It  is  as  follows: 

The  solution  is  freed  from  sulfuric  acid  by  neutralizing  to 
litmus  with  barium  hydroxide  and  adding  barium  nitrate  as  long  as 
a  precipitate  is  formed.  The  barium  sulfate  is  filtered  off  and 
washed.  The  solution  is  concentrated  to  300  cc.,  acidified  with 
nitric  acid,  if  necessary,  and  treated  with  silver  nitrate  until  an 
excess  of  silver  is  present.  The  solution  is  then  exactly  neutralized 
to  litmus  with  barium  hydroxide  and  5  cc.  of  a  cold  saturated  solu- 
tion of  barium  hydroxide  added.  If  10  cc.  of  the  filtered  solution, 
when  tested  with  a  drop  of  barium  hydroxide  solution,  give  a  pre- 
cipitate which  indicates  that  the  silver  salt  of  histidine  is  not  com- 
pletely thrown  down,  2  cc.  of  saturated  barium  hydroxide  solution 
are  added  to  the  main  bulk,  and  this  test  is  repeated  until  a  test  por- 
tion remains  clear.  The  precipitate  of  the  silver  salt  of  histidine 
is  then  filtered  off  and  washed  with  barium  hydroxide  until  free 
from  nitric  acid  and  the  filtrate  and  washings  treated  to  obtain 
arginine. 

It  was  found  difficult  to  determine,  by  the  above  method,  when 
the  silver  salt  of  histidine  had  been  completely  thrown  down. 

(b)  Kossel  and  Pringle2  tried  to  make  this  precipitation  more 
definite  by  adding  a  suspension  of  barium  carbonate  instead  of 

*Z.  physiol.  Chem.   (1900-1901),  31;  165-214. 
2Z.  physiol.  Chem.  (1906),  49;  301-324. 


excess  of  barium  hydroxide  to  the  neutral  solution  obtained  above, 
warming  the  suspension  on  a  water  bath,  and  then  raising  it  to  the 
boiling  point.  After  cooling,  the  histidine  silver  compound  is  fil- 
tered off  and  washed. 

No  advantage  was  found  in  this  method  and  it  seems  to  afford 
more  chance  for  decomposition  to  take  place. 

(c)  The  modification  recommended  by  Osborne,  Leavenworth 
and  Brautlecht1  was  found  to  be  the  most  definite.  In  it  the  greater 
portion  of  the  histidine  is  first  removed  from  the  above  mixture  by 
precipitation  with  mercuric  sulfate.  It  is  then  reprecipitated  as  the 
silver  compound. 

The  filtrate  from  the  mercury  precipitate  still  contains  a  small 
amount  of  histidine  because  of  the  solubility  of  the  mercury  histidine 
compound.  This  is  separated  as  the  silver  salt,  as  described  above, 
under  '(a)'.  The  work  was  carried  out  as  follows: 

The  solution  obtained  by  decomposing  the  silver  compound  of 
histidine  and  arginine  was  concentrated  to  about  250  cc.,  sulfuric 
acid  was  added  until  the  solution  contained  5  per  cent  of  this  acid, 
and  a  slight  excess  of  mercuric  sulfate  solution  then  added.  The 
precipitate  formed  was  allowed  to  stand  for  twelve  to  twenty-four 
hours,  was  then  filtered  off,  washed  with  5  per  cent  sulfuric  acid, 
suspended  in  water,  and  decomposed  with  hydrogen  sulfide.  The 
filtrate  and  washings  from  the  mercuric  sulfide  which  contained  the 
histidine  were  neutralized  with  barium  hydroxide  solution,  and 
barium  nitrate  was  added  until  no  more  barium  sulfate  was  precipi- 
tated. The  barium  sulfate  was  filtered  off  and  carefully  washed. 

The  histidine  was  then  thrown  down  again,  this  time  as  the 
silver  compound  as  described  under  '(a)'  above. 

The  filtrate  and  washings  from  the  mercury  histidine  precipi- 
tate which  contained  the  arginine  and  a  small  amount  of  histidine 
was  freed  from  mercury  with  hydrogen  sulfide,  freed  from  hydro- 
gen sulfide  by  evaporation,  neutralized,  and  treated  as  described 
under  '(a)'  to  throw  down  the  silver  histidine  compound.  This 
precipitation  was  found  to  be  quite  definite,  and  the  amount  of 
barium  hydroxide  solution  needed  was  small. 

Decomposition  of  the  Histidine  Silver  Compound. — The  com- 
bined precipitates  of  the  silver  compound  of  histidine  were  sus- 

.  Jour.  Physiol.  (1908),  23;  180-200. 


— 9— 

pended  in  water  made  acid  with  sulfuric  acid  and  decomposed  with 
hydrogen  sulfide.  The  silver  sulfide  was  filtered  off  and  carefully 
washed.  The  solutions  and  washings  were  concentrated  and  made 
up  to  a  definite  volume  so  that  nitrogen  determinations  could  be 
made. 

Preparation  of  Histidine  Solution  for  Use. — The  above  solu- 
tion was  made  alkaline  with  barium  hydroxide  and  the  barium  sul- 
fate  filtered  off  and  washed.  Excess  of  barium  was  then  removed 
by  treatment  with  carbon  dioxide  and  the  solution  evaporated  to 
dryness.  The  residue  was  extracted  with  hot  water,  made  up  to  a 
definite  volume  and  nitrogen  determinations  in  suitable  portions 
made.  The  amount  of  histidine  present  in  this  solution  was  then 
calculated.  It  was  found  to  be  1.66  per  cent  of  the  total  protein  for 
casein,  and  0.43  per  cent  of  the  total  protein  for  gelatin.  Osborne1 
reports  2.50  per  cent  of  the  total  protein  for  casein,  and  Hart2  gives 
0.40  per  cent  of  the  total  protein  for  gelatin. 

Decomposition  of  the  Arginine  Silver  Compound. — The  filtrate 
containing  the  arginine  was  saturated  with  finely  powdered  barium 
hydroxide  and  the  precipitate  of  the  arginine  silver  compound  so 
obtained  was  filtered  off  and  carefully  washed  by  stirring  up  in  a 
mortar  with  barium  hydroxide  and  filtering.  This  washing  was 
repeated  until  the  precipitate  was  free  from  nitric  acid.  The  pre- 
cipitate was  then  suspended  in  water  made  acid  with  sulfuric  acid 
and  decomposed  with  hydrogen  sulfide.  The  filtrate  and  washings 
from  the  silver  sulfide  and  barium  sulfate  were  evaporated  down 
to  a  definite  volume  so  that  nitrogen  determinations  could  be  made. 

Preparation  of  the  Arginine  Solution  for  Use. — The  above  so- 
lution was  freed  from  sulfuric  acid  with  barium  hydroxide  and 
treated  with  carbon  dioxide  until  a  precipitate  no  longer  formed. 
It  was  then  filtered,  evaporated  down  to  a  definite  volume  and  nitro- 
gen determinations  made.  From  these  the  amount  of  arginine 
present  was  calculated. 

The  amount  of  arginine  thus  calculated  was  found  to  be  7.50 
per  cent  of  the  total  protein  for  gelatin  and  3.40  per  cent  of  the 
total  protein  for  casein.  Kossel's3  figure  for  gelatin  is  9.3  per  cent, 
and  Osborne's4  for  casein  is  3.81  per  cent  of  the  total  protein. 

iJour.  Biol.  Chem.   (1911),  9;  333-353. 
2Hart,  Z.  physiol.  Chem.  (1901),  33;  358. 
3Z.  physiol.  Chem.  (1900-1901),  31;  204. 
*Jour.  Biol.  Chem.   (1911),  9;  333-353. 


—10— 

This  preparation  was  used  in  the  experiments  with  the  amylase 
because  the  separation  of  the  free  base  was  found  to  be  imprac- 
ticable and  the  compounds  which  could  be  prepared  in  crystalline 
form  would  introduce  substances  which  were  not  desired  in  the 
enzyme  work. 

EXPERIMENTS  WITH  THE  AMYLASE 

This  part  of  the  work  is  a  continuation  of  investigations  which 
have  been  carried  out  in  this  laboratory  on  the  influence  of  amino- 
acids  on  the  hydrolysis  of  starches  by  amylases.1  Since  previous 
work  had  shown  that  the  purified  preparations  of  pancreatic  amylase 
were  the  most  sensitive1  it  was  decided  to  study  one  of  these. 

Materials.  —  The  preparation  chosen  was  number  T-19-B, 
which  had  been  purified  in  this  laboratory  as  previously  described.2 

The  starch  was  Merck's  "soluble  starch  according  to  Lintner," 
washed  nine  times  with  ordinary  distilled  water  and  six  times  with 
triply  distilled  water.  Moisture  and  acidity  determinations  were 
made,  the  latter  by  careful  titration  with  0.0  IN  sodium  hydroxide, 
rosolic  acid  being  used  as  indicator. 

Ordinary  distilled  water  redistilled  twice,  (first  from  alkaline 
permanganate  solution  and  then  from  a  very  dilute  solution  of  phos- 
phoric acid  through  a  block  tin  condenser)  was  used  in  making  up 
all  solutions  and  starch  pastes,  and  for  the  final  rinsing  of  all 
glassware. 

Activating  solutions  were  made  up  with  sodium  chloride  and 
disodium  phosphate  which  had  been  recrystallized  twice  from  dis- 
tilled water  and  once  from  triply  distilled  water. 

Two  preparations  of  arginine  from  gelatin  and  three  prepara- 
tions of  histidine  (one  from  casein,  one  from  gelatin,  and  one  pur- 
chased from  the  Special  Chemicals  Company)  were  employed.  The 
cystine  had  been  prepared  by  another  worker  in  this  laboratory,3 
The  other  amino-acids  were  purchased  products,  glycine  (Eimer 
and  Amend),  phenylalanine  (Kahlbaum),  and  tryptophane  (Special 
Chemicals  Company  . 

Accurately  weighed  or  measured  portions  of  these  amino-acids 
were  dissolved  in  100  cc.  of  triply  distilled  water  and  titrated  with 


.  Am.  Chem.  Soc.  (1919),  41;  1866-1873,  and  unpublished  work. 
2Ibid  (1919),  41;  1855-1862. 
3Alice  Thompson  Merrill,  Dissertation  (1921). 


—11— 

0.01  N  sodium  hydroxide  or  0.01  N  hydrochloric  acid,  rosolic  acid 
being  used  as  indicator.  The  hydrogen-ion  concentrations  of  the 
starch  pastes  containing  the  neutralized  amino-acids  were  then  veri- 
fied by  electrometric  and  colorimetric  determinations. 

Method.  —  Preliminary  experiments  showed  that  the  gravimetric 
method  worked  out  in  this  laboratory  for  determining  the  saccharo- 
genic  power  of  the  enzyme1  could  not  be  used  in  the  presence  of 
arginine  and  histidine  because  of  their  interference  with  the  deter- 
mination of  reducing  sugar  by  Fehling's  solution.  This  had  also 
been  found  to  be  true  with  cystine.  For  this  reason  the  amyloclastic 
power  of  the  amylase  was  measured  instead. 

The  procedure  followed  was  based  on  the  method  of  Wohlge- 
muth,2  and  has  been  previously  used  and  described  in  this  labora- 
tory.3 Enough  starch  to  make  600  cc.  of  a  1  per  cent  starch  paste 
was  weighed  out,  mixed  with  50  cc.  of  triply  distilled  water  and 
poured  into  100  cc.  of  boiling  water.  The  paste  was  boiled  for 
three  minutes,  cooled,  and  25  cc.  poured  into  each  of  six  100  cc. 
cylinders.  To  each  of  these  were  added  the  proper  activating  agents 
(5  cc.  molar  sodium  chloride  and  2.5  cc.  M/50  disodium  phosphate), 
enough  0.0  IN  sodium  hydroxide  solution  to  neutralize  the  acidity 
of  the  starch,  and  the  amino-acid  to  be  tested,  which  was  also 
properly  neutralized.  The  mixture  in  each  cylinder  was  then  made 
up  to  100  cc.  with  triply  distilled  water  and  carefully  stirred. 

Forty-two  clean?  dry  test  tubes  were  placed  in  a  special  wire 
frame  basket  in  a  bath  of  ice  water  and  carefully  measured  por- 
tions of  enzyme  solution  introduced  into  each  by  means  of  a  1  cc. 
pipette  which  was  accurately  standardized  and  graduated  to  0.01  cc. 
Portions  of  5  cc.  of  one  of  the  starch  pastes  prepared  above  were 
then  introduced  into  each  of  seven  test  tubes.  This  was  done  care- 
fully by  means  of  a  burette  with  a  very  long  delivery  tip  reaching 
to  the  bottom  of  the  test  tube.  In  this  way  the  lodging  of  any  of 
the  starch  paste  on  the  sides  of  the  tube  was  avoided.  Since  the 
tubes  were  kept  in  ice  water  no  measurable  reaction  took  place. 

The  basket  of  tubes  thus  prepared  was  shaken  and  placed  in  a 
Freas  thermostat  in  which  the  temperature  varied  only  about 
±0.01  °C.  After  30  minutes  the  basket  was  taken  out  and  placed 
in  the  ice  water  to  stop  the  action.  The  tubes  were  thus  kept  at 


.  Am.  Chem.  Soc.   (1915),  37;  628. 
2Biochem.  Z.  (1908),  9;  1. 
"Jour.  Am.  Chem.  Soc.  (1915),  37;  634. 


—12— 

an  average  temperature  of  40°C.  for  30  minutes.  After  a  few 
minutes  0.1  cc.  of  0.1N  iodine  in  potassium  iodide  solution  was 
added  to  each  tube.  This  was  done  very  carefully  by  means  of  a 
dropping  bottle  which  delivers  drops  of  0.1  cc.  volume.  About  20  cc. 
of  distilled  water  was  then  poured  into  each  tube  and  the  contents 
thoroughly  mixed.  Each  set  of  tubes  containing  the  same  amount 
of  the  same  starch  paste  with  varying  amounts  of  enzyme  was  then 
observed  for  the  end  point;  that  is,  the  tube  of  lowest  enzyme 
concentration  which  is  definitely  red  and  shows  no  blue  or  violet 
color  due  to  starch.*  To  obtain  the  value  of  the  amyloclastic  power 
of  the  enzyme,  the  weight  of  the  1  per  cent  starch  paste,  5000  mg., 
is  divided  by  the  weight  in  milligrams  of  enzyme  present  in  the 
tube  showing  the  Wohlgemuth  end  point.  Blank  determinations 
were  always  made  in  each  set ;  that  is,  starch  pastes  neutralized  and 
containing  the  proper  activators  but  no  amino-acids  were  used  as 
the  standard  each  time,  since  results  vary  slightly  from  day  to  day 
due  to  experimental  error  and  deterioration  of  the  enzyme  in  solu- 
tion. 

Data  of  Typical  Experiments. — Table  I  shows  the  influence  of 
50  milligrams  of  arginine  and  50  milligrams  of  histidine  on  the 
amyloclastic  action  of  the  amylase.  It  will  be  seen  that  the  arginine 
has  a  distinct  "activating"  influence,  while  the  histidine  has  not. 
These  results  were  confirmed  by  other  experiments. 


*The  terms  used  in  describing  the  colors  are  those  of  the  Milton 
Bradley  Standard  Color  Chart  as  given  by  Mullikin  in  his  "Identification 
of  Pure  Organic  Compounds" 


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-18- 

In  order  to  prove  that  the  results  obtained  were  due  to  some 
influence  of  the  amino  acids  themselves  and  not  to  changes  in  the 
hydrogen-ion  concentration  of  the  digestion  mixtures,  determina- 
tions of  the  latter  were  made.  The  results  are  given  in  Table  VI 
and  show  that  a  uniform  hydrogen-ion  concentration  of  about 
CH+=lXlO~7  was  maintained  in  all  the  experiments.  This  con- 
centration had  been  found  by  previous  work  to  be  the  optimum  for 
the  saccharogenic  activity  of  the  amylase.1  In  some  cases  measure- 
ments of  the  hydrogen-ion  concentration  were  made  both  before  and 
after  digestion,  but  no  difference  was  found. 

Table  VI — Log  CH+  in  Moles  per  Liter  Found  in  Digestion 
Mixtures  Used.2 


Starch  Paste 

Log  CH+  (electrometric) 

Log  CH+(  color  imetric) 

No  amino  acid 

—6.94 

—6.96 

Arginine  VI,  50  mg. 

—6.92 

—6.96 

'  Arginine  V,  50  mg. 

—6.96 

Glycine,  50  mg. 

—6.93 

—6.96 

Histidine,  50  mg. 
(casein) 

—7.00 

—6.79  to  —6.96 

Histidine,  50  mg. 
(gelatin) 

—6.96 

Histidine,  50  mg. 
(purchased) 

—6.93 

—6.79  to  —6.96 

Tryptophane,  50  mg. 

—6.93 

—6.96 

Cystine,  50  mg. 

—6.79  to  —6.96 

Phenylalanine,  50  mg. 

—6.96 

Since  sodium  chloride  was  added  to  each  starch  paste  in  the 
amount  which  had  been  shown  to  give  the  optimum  saccharogenic 
activity,  the  results  obtained  were  not  due  to  changes  in  the  con- 
centration of  inorganic  salts. 


iJour.  Am.  Chem.  Soc.  (1919),  41;  231-235. 

2The  colorimetric  determinations  of  the  hydrogen-ion  concentrations 
were  made  with  Sorensen's  phosphate  buffer  mixtures,  as  described  by 
Clark,  as  standards,  ["Determination  of  Hydrogen-ions,"  W.  M.  Clark 
(1920),  76].  The  electrometric  determinations  were  made  with  the 
Clark  rocking  electrode,  [Jour.  Biol.  Chem.  (1915,  23;  475-486]. 


—19— 

The  possible  reaction  between  iodine  and  histidine  and  trypto- 
phane  was  considered.  It  was  shown  by  tests  with  larger  amounts 
of  iodine,  controlled  by  suitable  blanks,  that  this  was  not  the  reason 
for  the  difference  in  the  results  obtained  with  these  amino  acids. 

The  reason  for  the  difference  in  the  behavior  of  these  amino 
acids  as  compared  with  that  of  the  other  amino  acids  studied  must 
be  sought  in  either  or  both  of  two  ways:  in  the  influence  of  the 
structure  of  these  amino  acids  themselves,  some  inhibitory  influence 
of  their  heterocycles ;  or  in  the  structure  of  the  molecule  of  the 
protein  substance  which  makes  up,  or  is  essential  to,  the  enzyme. 

SUMMARY 

Preparations  of  arginine  and  histidine  for  use  in  the  study  of 
the  hydrolysis  of  "soluble"  starch  by  pancreatic  amylase  were 
separated  from  the  hydrolysis  products  of  casein  and  gelatin.  Kos- 
sel's  method  for  the  hydrolysis  of  protein  and  separation  of  the 
"diamino"  acids,  as  modified  by  other  workers  and  summarized  by 
Plimmer,  was  followed,  and  certain  suggestions  for  its  standardiza- 
tion made. 

Two  preparations  of  arginine  thus  separated  from  gelatin,  and 
two  of  histidine,  one  from  gelatin  and  the  other  from  casein,  were 
tested  for  their  influence  upon  the  action  of  the  enzyme  in  compari- 
son with  blank  tests,  with  the  monoamino  acids,  glycine  and  phe- 
nylalanine,  with  each  other,  with  purchased  preparations  of  histi- 
dine and  tryptophane,  and  with  cystine  prepared  by  another  worker 
in  this  laboratory. 

Tested  by  measuring  the  amyloclastic  action  of  the  purified 
pancreatic  amylase  upon  "soluble"  starch,  it  has  been  shown  that 
arginine  "activates"  the  digestion  of  the  starch  and  that  histidine 
does  not.  Consistent  results  were  obtained  with  the  two  prepara- 
tions of  arginine  and  the  three  of  histidine. 

Glycine,  phenylalanine  and  cystine  acted  like  arginine  in  "acti- 
vating" the  pancreatic  amylase. 

Tryptophane  resembled  histidine  in  showing  no  "activating" 
influence. 

The  tests  with  all  these  amino  acids  were  controlled  as  to 
hydrogen-ion  concentration,  in  most  cases  by  both  the  colorimetric 
and  the  electrometric  methods.  It  is  therefore  established  that  the 
influence  of  the  amino  acid  upon  the  extent  of  the  hydrolysis  of  the 


—20  — 

starch  by  the  enzyme  is  not  simply  a  matter  of  buffer  effect  or 
other  effect  upon  the  hydrogen-ion  concentration  of  the  digestion 
mixture. 

Since  all  the  amino  acids  here  considered  contain  the  a  amino 
group  to  which  must  be  attributed  an  "activating"  influence  (since 
this  is  the  feature  which  they  possess  in  common,  and  the  only  fea- 
ture to  which  the  "activating"  influence  of  glycine  can  be  ascribed) 
it  follows  that  the  negative  results  obtained  with  histidine  and  tryp- 
tophane  must  be  due  to  some  inhibitory  influence  exerted  by  their 
respective  heterocycles. 

The  "activation"  of  amyloclastic  action  by  glycine  and  phenyl- 
alanine,  here  established  for  the  first  time,  is  in  accordance  with 
their  "activation"  of  the  saccharogenic  power  of  the  same  enzyme 
as  previously  determined  in  this  laboratory. 

Cystine,  the  influence  of  which  upon  saccharogenic  action  could 
not  be  established  because  of  its  interference  with  the  determination 
of  reducing  sugar  by  Fehling's  solution,  is  here  shown  to  yield 
positive  results  when  studied  by  the  amyloclastic  method. 

The  influence  of  arginine,  histidine  and  tryptophane  upon  the 
activity  of  a  purified  enzyme  preparation  has  here  been  studied  for 
the  first  time,  and  it  has  been  shown  that  these  amino  acids  differ 
among  themselves  in  their  effects,  whereas  the  different  monoamino 
acids  studied  have  shown  a  practically  uniform  behavior  in  this 
respect. 


VITA 

Mary  Letitia  Caldwell  was  born  in  Bogota,  Colombia,  S.  America, 
December  18,  1890.  She  was  graduated  from  the  high  school  in 
Greenfield,  Ohio,  in  1909.  In  1913  she  received  the  degree  of 
Bachelor  of  Arts  from  Western  College  for  Women,  Oxford,  Ohio, 
where  she  held  the  position  of  Instructor  in  Chemistry  from  1914 
to  1918.  She  has  been  a  graduate  student  in  the  Faculty  of  Pure 
Science,  Columbia  University,  during  the  summers  of  1917,  1919, 
1920,  and  academic  years  1918-1919,  1919-1920  and  1920-1921,  re- 
ceiving the  degree  of  Master  of  Arts  in  1919.  She  has  held  a  Uni- 
versity Fellowship  for  the  year  1920-1921. 

She  was  co-author  with  H.  C.  Sherman  and  Florence  Walker 
of  a  paper  entitled  '"Action  of  Enzymes  upon  Starches  of  Different 
Origin,"  published  in  the  Journal  of  the  American  Chemical 
Society,  41;  1123-1129. 


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